1. Field of the Invention
The present invention relates to screening or testing for insulin like growth factors and their binding proteins and related molecules (xe2x80x9cIGF/IGFBPsxe2x80x9d). More specifically the present invention relates to a method for screening IGF/IGFBPs from whole blood spotted on a solid support. Additionally, the present invention relates to a test kit for screening or testing IGF/IGFBPs on the solid support.
2. Description of the Prior Art
Insulin-like growth factors (IGFs) belong to a family of peptides that share a high degree of structural homology with insulin. IGFs (IGF-I and IGF-II) have important mitogenic and anabolic actions that are mediated by their binding to specific high affinity cell surface receptors (Jones J I, et al. Endocrin Rev 1995; 16:3-34). IGF-I and IGF-II are produced in multiple tissues and are present in blood and other biological fluids in association with specific high affinity IGF binding proteins (IGFBPs). Six structurally homologous IGFBPs with distinct molecular size, hormonal control, and tissue expression have been identified (Holly J M P, et al. Growth Regul 1994,4(suppl I):20-30). Although the precise biological significant of the IGFBPs have not been clearly defined, they appear to play important roles in a complex system which modulates the bioavalability and functions of the IGFs. In addition, the ability of the IGFBPs to directly bind to cell surface receptors and trigger IGF-independent cellular functions have been recently reported (Cohen, P, et al. Curr Opin Pediatr 1994;6:462-7). As used herein, the term xe2x80x9cIGF/IGFBPsxe2x80x9d refers to the IGFs, to IGFBPs and to acid labile subunit (ALS).
It is now well established that IGF-I is the major mediator of the in-vivo growth-promoting actions of growth hormone (GH) (Daughaday W H, et al. Endocrin Rev 1989 68-91). The IGFs circulate mostly ( greater than 85%) bound to an approximately 150 kD ternary protein complex consisting of IGFBP-3, the major serum IGFBP, and a unique, leucine-rich, acid-labile subunit (ALS). Smaller proportions of circulating IGFs are associated with other IGFBPs and less than 1% of serum IGF-I has been estimated to exist in an unbound or xe2x80x9cfreexe2x80x9d form (Rosenfeld R G, et al Recent Prog Horm Res 1990; 46:99-163).
The clinical assessment of growth hormone status has been controversial, primarily due to the episodic nature of GH secretion, its relatively short circulating half-life and considerable variability in GH measurement by different assay methods (Lee P D K, et al. Pediatr Res 1990:27:45-51: Rosenfeld R G et al. J Clin Endocrinol Metab 1995;80:1532-40). Currently, clinical evaluation of GH sufficiency invariably involves multiple venous blood sampling for the determination of pituitary GH secretion in response to a number of physiological or pharmacological stimuli. Because of the reporter limitations of provocative GH testing which include arbitrary definition of diagnostic cut-off levels, and potential health risk and cost, alternative screening procedures have been sought. As blood IGF-I and IGFBP-3 (as well as ALS) levels are highly dependent on GH secretion, determination of their serum levels, individually or in combination, have been recently recognized as the most effective means in the evaluation of childhood GH deficiency (Rosenfeld R G, et al. J Clin Endocrinol Metab 1995;80:1532-40). Unlike growth hormone, the circulating levels of IGF-I and IGFBP-3 in the ternary molecular complex with ALS remain relatively constant, thus, allowing reliable determination of their concentrations in a single random specimen. More recently, measurement of IGFBP-2/IGF-I ratios have been reported to further enhance the diagnostic utility of IGF-I and IGFBP-3 measurement (Smith W J, et al J Clin Endocrinol Metab 1993;77:1294-99).
In children blood sampling by way of venipuncture has been problematic. Alternative procedure involves collection of blood by the less invasive and more convenient capillary puncture from the heel, finger or earlobe. Capillary blood dried on filter paper is a well established approach and has been successfully used in a number of large scale infant and population screening programs (Dussault Jh, et al. J Pediatr 1974;86:670-4; Augier D. et al. J Genet Hum 1985;33:325-36; Chanteau S, et al. Trans R Soc Trop Med Hyg 1989:83:414-6).
The procedure allows collection of a relatively small volume of blood and has been regulated (Blood collection on filter paper for neonatal programs - 2d ed.; Approved Standard NCCLS publications LA4-A2. Villanova, Pa., 1992) to confer all the advantages of a reliable and safe specimen transportation system at a significantly low cost. The latter considerations may assume greater importance for applications requiring sample transportation to distant central laboratories and when transportation of liquid specimens may not be feasible and/or practical. The use of blood spots to enhance the stability of somatomedin is disclosed in U.S. Pat. No. 4,277,249 (Broughton).
We have recently described development of highly specific and simple non-competitive ELISA for reliable determination of IGF-I (Khosravi M J, et al. Clin Chem 1996;42:1147-54), IGF-II (Diamandi A, et al. The Endocrine Soc 77th Ann Meeting 1995: P2-328), IGFBP-3 (Khosravi J, et al. Clin Chem 1996;S6:234), IGFBP-I (Khosravi, J, et al. Clin Chem 1996;S6 171) and ALS in serum and other physiological fluids. The high affinity antibodies incorporated in these immunoassays have been selected for lack of cross-reactivity or interference by the closely related peptides or binding protein. However, because of the conserved nature of the IGF/IGFBPs, the immunoassays developed for human applications may be also useful for the analyte determination in other species. This may be of particular interest in veterinary medicine and livestock management where determination and monitoring of the IGFs and their binding proteins could have significant diagnostic or predictive values.
We here describe the first report on application of whole blood collected on filter paper in the analysis of the IGF/IGFBPs of human and veterinary interest. We demonstrate by example that blood dried on filter paper is ideal for the analysis of the IGF/IGFBPs as these analytes express high degree of stability in dried format and can be readily released from the solid-support by treatment with an appropriate elution reagent. Due to a naturally high concentrations of these analytes, their analysis in serum typically require a 50- to 100-fold sample pre-dilution. This requirement is advantageous as similar dilution factors could be incorporated in dried blood spot extraction procedures, thus, allowing employment of the current serum assays for the analysis of the extracted blood samples.
These and other advantages of the present invention will become apparent from the following detailed description.
The present invention provides a method of determining concentrations of an insulin-like growth factor or acid labile subunit in an individual comprising the steps of collecting blood having an unknown concentration of insulin-like growth factor or acid labile subunit from a subject; applying said blood onto a solid support; extracting blood from said solid support with a pre-extraction buffer to form a blood extract; contacting said blood extract with an acidification buffer, followed by a neutralization buffer to form a neutralized extract; and testing said neutralized extract for the concentration of insulin-like growth factor or acid labile subunit. The insulin-like growth factor may be IGF-I or IGF-II. The blood may be collected by capillary puncture. The capillary puncture may be finger prick, thumb prick or ear lobe prick.
Also provided in the present invention is a method of determining concentrations of an insulin-like growth factor binding protein or acid labile subunit comprising the steps of collecting blood having an unknown concentration of insulin-like growth factor binding protein from a subject; applying said blood onto a solid support; extracting blood from said solid support with a buffer extract to form a blood extract; and testing said blood extract for the concentration of insulin-like growth factor binding protein or acid labile subunit. The insulin-like growth factor binding protein may be IGFBP-xe2x88x921, xe2x88x922, xe2x88x923, xe2x88x924, xe2x88x925 or xe2x88x926.
Also provided herein is a kit for measuring insulin-like growth factor, acid labile subunit, and/or insulin-like growth factor binding protein.
These and other advantages of the present invention will become apparent through the following detailed description.